Maintenance of glucose homeostasis is central to our health and its failure results in diabetes mellitus. Despite current treatment regimens of several insulin injections per day, blood glucose levels still fluctuate significantly in diabetic patients, making diabetes the sixth leading cause of death in the United States. Alternative approaches to insulin injections include attempts to develop a cell therapy for diabetes by producing insulin- secreting ?-cells from human embryonic stem cells (hESCs) cells or by reprogramming other cell types into ?- cells. However, despite some success to differentiate hESCs into insulin-expressing cells, these cells express multiple hormones and are not capable of reversing diabetes. The main bottleneck for generating true functional ?-cells from other cell sources is the paucity of knowledge of how ?-cells are specified. We observed that conditional, stable activation of the transcription factor Nkx6.1 in endocrine progenitors introduces a ?-cell fate bias and disfavors differentiation into non-?-cell islet cell types. Moreover, we found that Nkx6.1 is required to maintain expression of ?-cell-specific programs of gene expression in adult mice. Research under this proposal will (a) define the molecular cues that specify ?-cells and repress alternative endocrine cell fates, (b) directly test whether the Nkx6.1 can reprogram non-?-cell islet cells into ?-cells in vivo and (c) genetically dissect the gene regulatory network controlled by Nkx6.1 in the regulation of adult ?-cell fate maintenance, proliferation and function. Knowledge gained from these studies will help devise strategies to resolve mixed endocrine lineage patterns in hESC-derived endocrine cells and to reprogram other cell types into fully functional ?-cells. PUBLIC HEALTH RELEVANCE: The initial success of isolated islet transplants in diabetic patients has been dampened by the realization that the low number of human donor pancreases will preclude a wide clinical application. Human embryonic stem cells (hESCs) could offer an unlimited cell supply, but it is currently unknown how to instruct hESC-derived pancreatic precursors to generate insulin-producing beta-cells. This proposal will define the mechanisms whereby beta-cells are generated from their precursors in mice and will define pathways that are important for generating new beta-cells during adulthood.